1. Field of the Invention
The present invention relates to methods for determining cellulolytic enhancing activity of polypeptides.
2. Description of the Related Art
Cellulose is a polymer of the simple sugar glucose linked by beta-1,4-bonds. Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta-glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it to attack by cellobiohydrolases. Cellobiohydrolases sequentially release molecules of cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble beta-1,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobiose to glucose.
The conversion of lignocellulosic feedstocks into ethanol has the advantages of the ready availability of large amounts of feedstock, the desirability of avoiding burning or land filling the materials, and the cleanliness of the ethanol fuel. Wood, agricultural residues, herbaceous crops, and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and lignin. Once the lignocellulose is converted to fermentable sugars, e.g., glucose, the fermentable sugars can easily be fermented by yeast into ethanol or other fermentation products.
WO 2005/074647 discloses isolated polypeptides having cellulolytic enhancing activity and polynucleotides thereof from Thielavia terrestris. WO 2005/074656 discloses an isolated polypeptide having cellulolytic enhancing activity and the polynucleotide thereof from Thermoascus aurantiacus. WO 2007/089290 discloses an isolated polypeptide having cellulolytic enhancing activity and the polynucleotide thereof from Trichoderma reesei. Such polypeptides enhance the activity of cellulolytic enzyme compositions in the hydrolysis of lignocellulosic feedstocks.
Current methods for evaluating whether a polypeptide has cellulolytic enhancing activity involve direct addition to cellulase compositions in the hydrolysis of process relevant pretreated biomass substrates. This has the disadvantage of high sample usage, limited sensitivity (lower signal to noise), and potential for variable results due to lot-to-lot variation in pretreated biomass.
There is a need in the art for improved methods that allow the identification and quantification of proteins that can enhance the hydrolysis of cellulosic materials by cellulolytic enzyme compositions.
The present invention provides methods for determining cellulolytic enhancing activity of a polypeptide.